Conjugating biologically-active proteins to polymers has been suggested to improve one or more of the properties of such proteins. Improved properties provided by conjugation of a bioactive material to a polymer include increased circulating life, increased water solubility and/or reduced antigenicity, relative to the same bioactive material in non-conjugated form. For example, some of the initial concepts of coupling peptides or polypeptides, including proteins, to polyethylene glycol (PEG) and similar water-soluble polymers, are disclosed by Davis et al., in U.S. Pat. No. 4,179,337, the disclosure of which is incorporated herein by reference. Such conjugates are usually formed by reacting a biologically active material, such as a protein, with a several-fold molar excess of an activated polymer, i.e., a polymer having a terminal linking group, without regard to where the polymer will attach to the protein.
Biologically active proteins that were among the first to be so conjugated include, e.g., insulin and hemoglobin. These proteins contain several free nucleophilic amino attachment sites, such as alpha amino groups (preferably N-terminal), epsilon amino groups and histidine residues, allowing several polymers to be attached, without significant loss of biologic activity.
In some instances, however, the conjugation reaction encounters complications. For example, conducting a conjugation reaction using excessive amounts of activated polymers can result in biologically inactive conjugates. Such inactivation can be caused by any of a number of undesirable reactions, including, for example, the formation of a linker bond to the protein that results in steric or conformational hindrance of a protein motif required for biological activity. This problem can be difficult to avoid since the polymer and protein are typically joined in solution-based reactions and there is little that can be done to preselect the points of polymer attachment.
It has been suggested that nonspecific polymer binding may be minimized by pre-blocking active sites with reversible (removable) protective materials, such as pyridoxal phosphate, but the results have been inconsistent.
Interferons (hereinafter may be referred to as "IFNs") are a group of proteins which could benefit from improved polymer conjugation techniques. One IFN species of great therapeutic potential that would especially benefit from such polymer-conjugation is alpha-interferon (hereinafter may be referred to as ".alpha.-IFN"). In the past, several polymer-interferon conjugates have been suggested. None of the prior art teachings, however, is believed to have disclosed that it was possible to selectively cleave any linkages that reduce the activity of the conjugated interferon.
U.S. Pat. Nos. 4,766,106 and 4,917,888, describe, inter alia, .beta.-interferon-PEG conjugates. The conjugates described in the aforementioned patents use either an amide or ester linkage to join the methoxypolyethylene glycol to the interferon. Wide molar ratios of the polymer and interferon are disclosed, ranging from 0.1 to 1,000:1.
U.S. Pat. No. 4,894,226, discloses .beta.-interferon conjugated via an amide linkage to polyproline using a flexible spacer arm. Similarly, U.S. Pat. No. 5,281,698, incorporated herein by reference in its entirety, describes, inter alia, reacting interferons with a urethane-linkage forming activated polyethylene glycol using a most preferred ratio of the polymer to the protein of 5:1. Also, U.S. Pat. No. 5,382,657, discloses forming amide or urethane-linked interferon conjugates using a polymer-protein ratio of about 3:1. Although relatively low molar excesses of the activated polymer were disclosed in these patents, there is no teaching or suggestion of controlling the location of covalent attachments between the polymer and protein or, more importantly, how to avoid forming conjugates containing polymers attached in the area of the active site. Thus, there was no mention of subjecting the conjugates to further treatment in order to enhance or regain the bioactivity lost by the conjugation reaction.
European Patent Application bearing publication No. 0 236 987 describes reacting alpha and gamma interferons with high molar excesses of alkyl-imido ester-activated polyethylene glycols. European Patent Application bearing publication No. 0 510 356 describes conjugating alpha interferon with pyridinyl carbonyl and thiocarbonyl activated PEG. In both cases, however, the resultant conjugates included species containing a wide variety of pegylated species, including a substantial amount containing more than one polymer strand per interferon molecule.
In other previous attempts to avoid loss of bioactivity following polymer conjugation, a granulocyte colony stimulating factor ("G-CSF")-polymer conjugate has been prepared by reacting G-CSF with a methoxy PEG carboxymethyl-N-hydroxy succinimidyl ester. The resulting conjugate is then treated with two molar hydroxylamine (pH 7.3) to remove "unstable" linkers, followed by a pH reduction to 3.5. Kinstler et al., 1996, Pharmaceutical Res. 13(7): 996-1002. The authors, however, provided no description or suggestion of attaining improved G-CSF nor have they provided any guidance regarding treatment of any other conjugates.
WO96/11953 reports that conjugates were prepared by reacting a protein, exemplified by consensus IFN, with a polymer, at an acid pH (pH 4). WO96/11953 states that this reaction selectively prevents linkage to lysine epsilon amino groups, while favoring linkage with the N-terminal alpha amino group. WO96/11953 also describes a two-step pH treatment process wherein G-CSF is reacted with a PEG at pH 8.0, followed by reduction of pH to pH 4.0, simply as a prelude to loading the product onto a separation column. No conjugates of interferon were reported to be prepared by this second method. In addition, it should be noted that WO96/11953 describes the use of an reductive alkylation reaction as preferred for the selective attachment of polymer, e.g., PEG, to the N-terminal and does not teach or suggest the advantages of an acylation reaction to attach polymers to IFN residues other than the N-terminal.
Therefore, it is believed that neither of the aforementioned Kinstler et al. references teaches or suggests preferentially removing a bioactivity-inhibiting linker bond from an active site of interferon.
In spite of the above-mentioned work in the area of interferon-polymer conjugates, improvements have been sought. In particular, it would be beneficial to provide interferon conjugates having substantially predictable and even uniform levels of bioactivity. It would also be beneficial to prepare IFN conjugates which are substantially free of polymers attached to the active site region of the interferon.
If an effective method of achieving polymer conjugation, while avoiding interference with alpha interferon bioactive sites were available, many useful .alpha.-IFN polymer conjugates could be prepared. Thus, a solution to the above-described problems would make optimally bioactive conjugates of alpha-interferon available to the art.